Bret assay

ABSTRACT

An improved BRET assay, wherein the BRET signal is enhanced and/or prolonged. The improved BRET assay comprises the steps of i) adding a substrate to a cell comprising GPCR-Rluc fusion protein and a β-arrestin-GFP fusion protein, wherein the (β-arrestin is mutated, ii) adding a ligand to obtain, if possible, a GPCR-Rluc/(β-arrestin-GFP complex, and iii) measuring a BRET signal to obtain a BRET ratio, wherein the improvement leads to an increased BRET ratio compared with the ratios obtained by use of the same process employing a β-arrestin-GFP fusion protein wherein the β-arrestin is the wild type β-arrestin, or employing a 13-arrestin-GFP fusion protein, wherein the (β-arrestin is a β-arrestin specifically mutated so that it acts on the receptor independent of the receptors phosphorylation state. The invention further relates to a stable substrate solution for use in an improved BRET assay.

FIELD OF THE INVENTION

The present invention relates to an improved BRET assay, wherein theBRET signal is enhanced and/or prolonged. The improved BRET assaycomprises the steps of

i) adding a substrate to a cell comprising GPCR-Rluc fusion protein anda p-arrestin-GFP fusion protein, wherein the β-arrestin is mutated,

ii) adding a ligand to obtain, if possible, a GPCR-Rluc/β-arrestin-GFPcomplex, and

iii) measuring a BRET signal to obtain a BRET ratio.

The invention further relates to a stable substrate solution for use inan improved BRET assay.

BACKGROUND OF THE INVENTION

BRET Assay

BRET (Bioluminescence Resonance Energy Transfer) assay is aprotein-protein interaction assay. It is based on energy transfer from abioluminescent donor to a fluorescent acceptor protein. This technologyuses a Renilla luciferase (Rluc) as the donor and a Green FluorescentProtein (GFP) as the acceptor molecule.

Rluc emits blue light (e.g. at 400 nm) in presence of its substrate. Ifa GFP molecule is in close proximity to the Rluc, it absorbs the bluelight energy and re-emits green light (e.g. at 515 nm). The BRET signal,therefore, is measured by the amount of green light emitted by GFP ascompared to the blue light emitted by Rluc. The ratio of green to blueincreases as the two proteins are brought into proximity. BRET assaysare performed by genetically fusing Rluc and GFP to biological partnersthat are expected to interact in a cell-based assay.

However, in certain situations where a GPCR-arrestin based assay isused, the BRET signal is relatively weak and short termed. Thus, thereis a need for improving the BRET assay in order to obtain a prolongedand/or enhanced signal.

DETAILED DISCLOSURE OF THE INVENTION

Accordingly, the present invention provides an improved BRET assay thatcomprises the following steps:

i) adding a substrate to a cell comprising GPCR-Rluc fusion protein anda β-arrestin-GFP fusion protein, wherein the β-arrestin is mutated,

ii) adding a ligand to obtain, if possible, a GPCR-Rluc/β-arrestin-GFPcomplex, and

iii) measuring a BRET signal to obtain a BRET ratio,

wherein the improvement leads to an increased BRET ratio compared withthe ratios obtained by use of the same process employing a βarrestin-GFP fusion protein, wherein the β-arrestin is the wild typeβ-arrestin or employing a β-arrestin-GFP fusion protein, wherein theβ-arrestin is a β-arrestin is specifically mutated so that it acts onthe receptor independent of the receptors phosphorylation state.

G Protein-Coupled Receptors for Use in the Present Invention

The G protein-coupled receptors (GPCRs) constitute the largest family ofproteins in the human genome and function as receivers of all kinds ofchemical signals. The spectrum of hormones, neurotransmitters, paracrinemediators etc., which act through G-protein coupled receptors includesall kinds of chemical messengers: Ions (calcium ions acting on theparathyroid and kidney chemosensor), amino acids (glutamate and -aminobutyric acid-GABA), monoamines (catecholamines, acetylcholine,serotonin, etc.), lipid messengers (prostaglandins, thromboxane,anandamide, (endogenous cannabinoid), platelet activating factor, etc.),purines (adenosine and ATP), neuropeptides (tachykinins, neuropeptide Y,endogenous opioids, cholecystokinin, vasoactive intestinal polypeptide(VIP), plus many others), peptide hormones (angiotensin, bradykinin,glucagon, calcitonin, parathyroid hormone, etc.), chemokines(interleukin-8, RANTES, MIP-1alpha etc.), glycoprotein hormones (TSH,LH/FSH, choriongonadotropin, etc.), as well as proteases (thrombin). Inour sensory systems, G-protein coupled receptors are involved both asthe light sensing molecules in the eye, i.e. rhodopsin and the colorpigment proteins, and as several hundreds of distinct odorant receptorsin the olfactory system as well as a large number of taste receptors.Structurally, G protein coupled receptors (GPCRs) are characterized byseven hydrophobic helical transmembrane segments connected by intra- andextracellular loops and are accordingly often referred to as 7TMreceptors.

Examples of 7TM receptors are the receptors for (—in brachet thereceptor subtypes are mentioned): acetylcholine (m1-5), adenosine (A1-3)and other purines and purimidines (P2U and P2Y1-12), adrenalin andnoradrenalin (α1A-D, α2A-D and β1-3), amylin, adrenomedullin,anaphylatoxin chemotactic factor, angiotensin (AT1A, −1B and −2),apelin, bombesin, bradykinin (1 and 2), C3a, C5a, calcitonin, calcitoningene related peptide, CD97, conopressin, corticotropin releasing factor(CRF1 and −2), calcium, cannabinoid (CB1 and −2), chemokines (CCR1-11,CXCR1-6, CX3CR and XCR), cholecystokinin (A-B), corticotropin-releasingfactor (CRF1-2), dopamine (D1-5), eicosanoids, endothelin (A and B),fMLP, Frizzled (Fz1,2,4,5 and 7-9), GABA (B1 and B2), galanin, gastrin,gastric inhibitory peptide, glucagon, glucagon-like peptide I and II,glutamate (1-8), glycoprotein hormone (e.g. FSH, LSH, TSH, LH), growthhormone releasing hormone, growth hormone secretagogue/Ghrelin,histamine (H1-4), 5-hydroxytryptamine (5HT1A-1F, −2A-C and −4-7),leukotriene, lysophospholipid (EDG1-4), melanocortins (MC1-5), melaninconcentrating hormone (MCH 1 and 2), melatonin (ML1A and 1B), motilin,neuromedin U, neuropeptide FF (NFF1 and 2), neuropeptide Y (NPY1,2,4,5and 6), neurotensin (1 and 2), nocioceptin, odor components, opiods (κ,δ, μ and x), orexins(OX1 and −2), oxytocin, parathyroidhormone/parathyroid hormone-related peptides, pheromones,platelet-activating factor, prostaglandin (EP1-4 and F2) prostacyclin,pituitary adenylate activating peptide, retinal, secretin, smoothernd,somatostatins (SSTR1-5), tachykinins (NK1-3), thrombin and otherproteases acting through 7TM receptor, thromboxane,thyrotropin-releasing hormone, vasopressin (V1A, −1B and −2), vasoactiveintestinal peptide, urotensin II, and virally encoded receptors (US27,US28, UL33, UL78, ORF74, U12, U51); and 7TM proteins coded for in thehuman genome but for which no endogenous ligand has yet been assignedsuch as mas-proto-oncogene, EBI (I and II), lactrophilin, brain specificangiogenesis inhibitor (BAI1-3), EMR1, RDC1 receptor, GPR12 receptor orGPR3 receptor, and 7TM proteins coded for in the human genome but forwhich no endogenous ligand has yet been assigned.

Arrestins and GRKs Role in Receptor Signalling

Arrestins and GRKs (G-protein coupled receptor kinases) play importantroles in the regulation of 7TM receptor responsiveness by terminatingthe G protein mediated signal. After agonist binding to a GPCR, thereceptor changes conformation and is then phosphorylated by a GRK.Arrestins will then translocate to the phosphorylated receptor and bindto the receptor within seconds or minutes after agonist stimulation.Full inactivation of the G-protein mediated 7TM receptor signaling isachieved through binding of one of a family of arrestin molecules, whichsterically hinder G protein binding.

Arrestin functions as an adaptor protein, which will connect thereceptor to clathrin and AP-2 in clathrin coated pits, which results insequestration of the receptor into intracellular vesicles of theendosomal pathway in which dynamin plays an important role in the actualvesicular sequestration process. The mechanisms involved in thetransport of the arrestin-receptor complex to the clathrin coated pit isnot fully understood, but it is becoming clear that the binding ofarrestin to parts of the cell membrane e.g. to phosphoinositides isessential.

The family of arrestins has at least four members showing a high degreeof amino acid homology and classified primarily on the basis of tissuedistribution. They include (i) visual arrestin and (ii) C-arrestin,which are mostly restricted to the eye, and the non-visual-arrestins(iii) β-arrestin 1 and (iv) β-arrestin 2, distributed ubiquitously inalmost every tissue. β-arrestins share more than 70% amino acididentity.

Arrestins are composed of three structural and functional parts, anamino-terminal domain, which binds to the receptor, a carboxyl-terminaldomain, which connects to proteins involved in receptor-sequestration,such as clathrin and AP-2 (adaptor protein 2) and a central part whichconnects to components of the cell membrane, such as phosphoinositides.Visual arrestins, which mainly interacts with the rhodopsin receptor,are very weak in their clathrin-association and are in general notconsidered to be capable of mediating receptor internalisation.

As described above arrestin are translocated to the activated andphosphorylated GPCR within minutes after agonist simulation. Thisinteraction is universal for almost all GPCRs upon activation. Thus, aBRET assay based on the GPCR-arrestin interaction, wherein the GPCR isfused with Rluc and arrestin is fused with GFP, is a very useful assayfor a wide range of receptors, and it also provides means for thediscovery of ligands that interact with GPCRs of unknown function i.e.orphan GPCRs.

However, as described above, arrestin functions as an adaptor protein,which will connect the receptor to clathrin and AP-2, which results insequestration of the receptor into intracellular vesicles. Afterinternalization of the receptor/arrestin complex, arrestin willdissociate from the receptor and the BRET signal is terminated. Thedissociation kinetics can be fast or slow depending on the receptortype. For Class A type receptors, the dissociation is usually fast,whereas for Class B type receptors the dissociation is slower.

As mentioned above, β-arrestin associates to a receptor after thereceptor has been phosphorylated by a GRK.

GRKs are 57-80 kDa proteins that are members of the large family ofserine/threonine kinases. The human GRKs are part of a family of atleast 7 GRKs (GRK1-7). Some of the GRKs have been shown to be expressedin a tissue specific manner, i.e. GRK1 (rhodopsin kinase), which isexpressed in retina, and GRK4, which is expressed in testis. Incontrast, the other GRKs are more widely distributed and evidence existsto suggest that these GRKs are likely to be involved in desensitizationof multiple types of GPCRs.

Definitions

Throughout the text including the claims, the following terms shall bedefined as indicated below.

In the present context the term “BRET ratio” is intended to mean theratio of green light emitted by GFP as compared to the blue lightemitted by Rluc. In the present context the terms “BRET signal” and“BRET ratio” are intended to have the same meaning.

A “ligand” is intended to include a substance that either inhibits orstimulates the activity of a receptor and/or that competes for thereceptor in a binding assay. An “agonist” is defined as a ligandincreasing the functional activity of a biological target molecule. An“antagonist” is defined as a ligand decreasing the functional activityof a biological target molecule either by inhibiting the action of anagonist or by its own intrinsic activity. An “inverse agonist” (alsotermed “negative antagonist”) is defined as a ligand decreasing thebasal functional activity of a biological target molecule

In the present context the term “improved BRET assay” denotes an assaywhere the BRET ratio is increased by at least about 5% such as, e.g., atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about65%, at least about 70%, at least about 75%, at least about 80%, ormore.

In the present context, the term “β-arrestin that is specificallymutated so that it acts on the receptor independent of the receptorsphosphorylation state” means that β-arrestin is mutated deliberatelywith the purpose of becoming phosphorylation independent. An example ofsuch a mutation is R169E human β-arrestin-2, wherein arginine has beenchanged to glutamic acid. β-arrestins being mutated for other purposes,but accidentally also being phosphorylation independent, are notencompassed in this definition.

As described above, the present invention describes an improved BRETassay wherein the BRET signal is enhanced and/or prolonged.

Since the BRET signal is dependent on the association/dissociation ofthe GPCR-Rluc/β-arrestin-GFP complex, prevention of the dissociation ofthe complex will enhance and/or prolong the BRET signal.

Thus, the invention also provides an improved assay, wherein theseparation of β-arrestin-GFP from GPCR-Rluc/β-arrestin-GFP complex isdelayed and/or inhibited.

As described above, the GPCR-Rluc/β-arrestin-GFP complex dissociateswhen the complex is internalized. Thus, inhibition of theinternalization will prevent dissociation and accordingly, the BRETsignal will be enhanced and/or prolonged. Accordingly, the inventionalso relates to an improved assay wherein the internalization ofGPCR-Rluc/β-arrestin-GFP complex is inhibited.

One way of inhibiting internalization is to prevent the binding ofβ-arrestin to clathrin and AP-2. In an improved assay according to theinvention, β-arrestin is mutated so that its binding to clathrin and/orAP2 is impaired.

As described above, the carboxyl-terminal domain of arrestins connectsto proteins involved in receptor-sequestration, such as clathrin andAP-2. Accordingly, in one assay according to the invention, theβ-arrestin is truncated so that it does not contain any clathrin and/orAP2 binding sites, i.e. a part of the β-arrestin from the C-terminal endhas been deleted.

In one assay according to the invention β-arrestin is mutated bydeletion, insertion or substitution so that one or more AP2 bindingsites of β-arrestin are impaired in their binding to AP2.

Specific examples of a truncated β-arrestin are a human β-arrestin-1 374stop mutant or human β-arrestin-2 373 stop mutant.

A specific example of a β-arrestin mutated by substitution is the humanβ-arrestin-2 R393E; R395E mutant, wherein the amino acids number 393 and395 have been changed from arginine to glutamic acid. Another example ofa β-arrestin mutated by substitution is the human β-arrestin-2 R393A;R395A mutant, wherein the amino acids number 393 and 395 have beenchanged from arginine to alanine. Other substitutions may of course beused.

As described above, there are at least 4 family members of arrestins.Furthermore, the arrestins are found in animals including rodents,swine, poultry, cattle, sheep, goats, horses, cats, dogs, monkeys andhumans. Thus, the specific mutations mentioned only tend as illustrativeexamples. All arrestins are usable in an assay according to theinvention, and the specific position of the truncation and othermutations will depend on the species and type of arrestin. As anexample, to impair the AP2 binding sites in bovine β-arrestin-2 aminoacids number 394 and 396 should be substituted from arginine to glutamicacid as compared to the R393E and R395E substitutions in humanβ-arrestin.

Another way of inhibiting the internalization of theGPCR-Rluc/β-arrestin-GFP complex is to prevent the binding of β-arrestinto phosphoinositide. By impairing the binding of β-arrestin tophosphoinositide, the transport of the GPCR-Rluc/β-arrestin-GFP complexto the clathrin coated pits is impaired.

In order to impair binding to phosphoinositide β-arrestin may be mutatedin any suitable way e.g. by deletion, insertion or substitution.

A specific example of a β-arrestin mutant, wherein the binding tophosphoinositide is impaired is the triple mutant human β-arrestin-2K233Q; R237Q; K251Q, wherein amino acid no. 233 has been changed fromlysine to glutamine, amino acid no. 237 has been changed from arginineto glutamine and amino acid no. 251 has been changed from lysine toglutamine.

The examples given above are all related to ways of improving/prolongingthe BRET signal by prevention of the dissociation of theGPCR-Rluc/β-arrestin-GFP complex

The invention also provides an improved assay, wherein the associationof β-arrestin-GFP to the GPCR is increased, which will in turn increasethe BRET signal.

As described above, β-arrestin associates to a receptor after thereceptor has been phosphorylated by a GRK. In some cases the receptorused in an assay as described herein will be phosphorylated slowlyand/or not fully, i.e. if the amount of GRK naturally present in thecells is low. This results in slow and/or reduced binding of β-arrestinto the receptor, leading to a reduced BRET signal. Thus, by increasingthe phosphorylation of the receptor the BRET signal will be enhanced. Asthe phosphorylation of a GPCR by a GRK is a virtually universal event, aGPCR-Rluc/β-arrestin-GFP based assay as described above, further aidedby the addition of a GRK is a very useful assay, especially if thephosphorylation of the GPCR is rate limiting step in the association ofGPCR-Rluc and β-arrestin-GFP.

Accordingly, the invention also relates to an improved assay, whereinthe cell comprises a further amount of G-protein coupled receptor kinaseas compared to the amount of GRK naturally present in the cell.

In a specific example, the G-protein coupled receptor kinase is GRK 2 orGRK-5.

As described above, there are at least 7 family members of human GRKs.Furthermore, the GRKs are found in other animals including rodents,swine, poultry, cattle, sheep, goats, horses, cats, dogs and monkeys.Thus, the specific GRKs mentioned only tend as illustrative examples asall GRKs are usable in an assay according to the invention.

The β-arrestin may be further mutated so that it, besides havingimpaired binding capability to clathrin, AP2 and/or phosphoinositide,also is phosphorylation independent. As described above, the binding ofβ-arrestin to the GPCR requires the phosphorylation of the receptor, thelatter being a rate limiting step in some cases. By using aphosphorylation independent β-arrestin mutant in an assay according tothe invention, the degree of phosphorylation of the receptor will nolonger have any influence on the formation of theGPCR-Rluc/β-arrestin-GFP complex, leading to a higher BRET signal incases, where phosphorylation of the receptor is a rate limiting step.

The invention also relates to all suitable combinations of β-arrestinmutants, i.e, a mutant having impaired binding capability to bothclathrin and/or AP2 and phosphoinositide.

ASPECTS OF THE INVENTION

The improved BRET assay may be used in drug discovery methods, such asscreening assays for identifying new ligands of GCPRs. The BRET assaymay also be used for the discovery of ligands that interact with GPCRsof unknown function i.e. orphan GPCRs.

The ligands may be agonists or antagonists. If the ligand is a knownantagonist, or if the assay is set up to screen for unknown antagonists,the improved BRET assay further comprises the addition of an agonistafter adding the antagonist, or suspected antagonist ligand.

The invention also relates to an improved assay according to theinvention for use in high-throughput screening.

One of the problems associated with setting up an assay, such as, e.g. ahigh through-put screening (HTS) assay, is to prepare a suitablesolution of the substrate, DeepBlueC™. If the stock solution ofDeepBlueC™ is diluted to the relevant concentration for use in a BRETassay in D-PBS as recommended, it will form a precipitate within 10 to20 minutes, making it virtually impossible to use for a HTS assay.

The present inventors have found that a solution of DeepBlueC™ in aproper amount of organic solvent prevents the formation of precipitate.Accordingly, the present invention relates to a solution comprisingDeepBlueC™ and one or more organic solvents, wherein no visualprecipitate is formed after storage at room temperature for at least 30min such as, e.g., at least about 45 min, for at least about 1 hr, forat least about 1.5 hrs, for at least about 2 hrs, for at least about 2.5hrs, for at least about 3 hrs, for at least about 3.5 hrs or for atleast about 4 hrs.

The organic solvent may be chosen from any organic solvent usable in aBRET assay, such as, e.g. an alkanol including ethanol, propanol,isopropanol, and butanol. In a specific embodiment, the solvent isethanol.

As illustrated in the examples, the formation of precipitate isinterrelated to the concentration of the organic solvent in thesolution.

When the concentration of organic solvent is decreased from a optimallevel, the amount of precipitate will increase, and the time beforeformation of precipitate will decrease. On the other hand, if theconcentration of organic solvent in the solution is too high it may notbe suitable for use in a BRET assay, as the BRET signal will decrease ifthe percentage of organic solvent in the assay gets too high.

The invention relates to a solution, comprising from about 15% v/v EtOHto about 100 v/v % EtOH, such as, e.g. from about 20% v/v EtOH to about90% v/v EtOH, from about 30% v/v EtOH to about 80% v/v EtOH, from about35% v/v EtOH to about 75% v/v EtOH, from about 35% v/v EtOH to about 70%v/v EtOH, from about 35% v/v EtOH to about 65% v/v EtOH, from about 35%v/v EtOH to about 60% v/v EtOH, from about 40% v/v EtOH to about 60% v/vEtOH, from about 40% v/v EtOH to about 55% v/v EtOH or from about 40%v/v EtOH to about 50% v/v EtOH.

In a specific embodiment, the invention relates to a solution,comprising DeepBlueC™ in 40% v/v EtOH.

The invention also relates to a solution, further comprising D-PBS.

The invention also relates to a method for preparing a solution asdescribed above, the method comprising diluting a stock solution ofDeepBlueC™ in a solution comprising one or more organic solvents. Theactual dilution of DeepBlueC™ depends on the conditions of the specificassay, wherein the solution is to be used. A specific example of asuitable solution appears from the Examples, without limiting theinvention hereto.

The substrate solution is suitable for use in an improved BRET assay asdescribed herein. I.e., the invention relates to an improved assayaccording to any of the preceding claims, wherein the substrate is usedin the form of a solution from which no visual precipitate is formedafter storage at room temperature for at least 30 min such as, e.g., forat least about 45 min, for at least about 1 hr, for at least about 1.5hrs, for at least about 2 hrs, for at least about 2.5 hrs, for at leastabout 3 hrs, for at least about 3.5 hrs or for at least about 4 hrs.

The invention also relates to an improved assay, wherein the solutioncontaining the substrate comprises one or more organic solvents. The oneor more organic solvents may be selected from alkanols includingethanol, propanol, isopropanol, and butanol. In a specific embodiment,the solvent is EtOH.

The invention further relates to an improved assay, wherein the solutioncomprises from about 15% v/v EtOH to about 100 v/v % EtOH, such as, e.g.from about 20% v/v EtOH to about 90% v/v EtOH, from about 30% v/v EtOHto about 80% v/v EtOH, from about 35% v/v EtOH to about 75% v/v EtOH,from about 35% v/v EtOH to about 70% v/v EtOH, from about 35% v/v EtOHto about 65% v/v EtOH, from about 35% v/v EtOH to about 60% v/v EtOH,from about 40% v/v EtOH to about 60% v/v EtOH, from about 40% v/v EtOHto about 55% v/v EtOH or from about 40% v/v EtOH to about 50% v/v EtOH.

In a specific embodiment, the solution comprises DeepBlueC™ in 40% v/vEtOH.

OTHER ASPECTS OF THE INVENTION

Other aspects of the invention appear from the appended claims. Thedetails and particulars described above and relating to the methodaccording to the invention apply mutatis mutandis to the other aspectsof the invention.

LEGENDS TO FIGURES

FIG. 1 shows internalization of the NK1 and the β2AR receptorsco-expressed in cells together with WT or one of the three differentβ-arrestin mutants: human β-arrestin-2 R393E; R395E mutant, humanβ-arrestin-2 373 stop mutant or human β-arrestin-2 R169E mutant.

FIG. 2 shows the specific BRET ratio of the NK1 and the β2AR receptorsco-expressed with WT or one of the three different β-arrestin mutants:human β-arrestin-2 R393E; R395E mutant, human β-arrestin-2 373 stopmutant or human β-arrestin-2 R169E mutant.

FIG. 3 shows the light emission from renilla luciferase after additionof DeepBlueC dissolved in buffer (D-PBS with 1000 mg/l L-Glucose),buffer with 6% ethanol or buffer with 40% ethanol.

The following examples are intended to illustrate the invention withoutlimiting it thereto.

EXAMPLES

NK-1 Receptor Internalization Assays

COS7 cells in 75 cm² flask (3×10⁶ cells/flask) were used fortransfection. NK-1/Rluc receptor (2 μg cDNA/flask) was coexpressedtogether with 6 μg GFP/β-arrestin 2, 6 μg GFP²/β-arrestin R169E, 6 μgGFP²/β-arrestin Lys 373 stop or 6 μg GFP²/β-arrestin R393E, R395E. Atthe end of transfection period (3-5 hours), cells were washed twice withPBS, trypsinased and plated at a density of 2.5×10⁵ cells per well in12-well plates. After 48 hours, cells were washed once with assay medium(HEPES-modified DMEM with 0.1% BSA, pH 7.4) and incubated in assaymedium for at least 1 hour before being incubated with ¹²⁵I-labeled SP(30000 cpm/well) in 0.5 ml assay medium 10 min at 37 C. Cells were thentransferred onto ice and washed twice with ice-cold PBS. Subsequently,the extracellular receptor-associated ligand was removed by washing oncewith 1 ml of acid solution (50 mM acetic acid and 150 mM NaCl, pH 2.8)for 12 min. The acid wash was collected to determine the surface-boundradioactivity, and the internalized radioactivity was determined aftersolubilizing the cells in 0.2 M NaOH and 1% SDS (NaOH/SDS) solution.Nonspecific binding for each time point was determined under the sameconditions in the presence of 1 μM unlabeled agonist (SP). Aftersubtraction of nonspecific binding, the internalized radioactivity wasexpressed as a percentage of the total binding.

FIG. 1 shows the internalization of the NK1-R co-expressed in cellstogether with WT or one of the three different β-arrestin mutants. Thefigure illustrates that the human β-arrestin-2 R393E; R395E mutant andthe human β-arrestin-2 373 stop mutant are inhibiting theinternalization.

β2AR Internalization Assays

COS-7 cells in 75 cm² flask (3×10⁶ cells/flask) were used fortrasfection. β₂AR/Rluc receptor (1.3 μg cDNA/flask) was coexpressedtogether with 6.5 μg GFP²/β-arrestin 2, 6.5 μg GFP²/β-arrestin R169E,6.5 μg GFP²/β-arrestin Lys 373 stop or 6.5 μg GFP²/β-arrestin R393E,R395E. Receptor internalization assay was based on protocol described byBarak and Caron J Recept Signal Transduct Res 1995January-March;15(14):677-90. At the end of transfection period (3-5hours), cells were washed twice with PBS, trypsinesed and plated at adensity of 2.5×10⁵ cells per well in 12-well plates. After 48 hours,cells were washed once with assay medium (HEPES-modified DMEM with 0.1%BSA, pH 7.4) and serum-starved in the same medium for additional 2-3hours before being stimulated with 1 mM isopterenol for 10 min at 37° C.Stimulation was stopped by washing the cells with ice-cold PBS. Cellswere then subjected to [¹²⁵I]-pindolol binding at 4° C. for 3 h and thefraction of internalized receptors determined relative to unstimulatedcells. Non-specific binding was determined under the same conditions inthe presence of 1 μM pindolol.

FIG. 1 shows the internalization of the β2AR co-expressed in cellstogether with Wt or one of the three different β-arrestin mutants. Thefigure illustrates that the human β-arrestin-2 R393E; R395E mutant andthe human β-arrestin-2 373 stop mutant are inhibiting theinternalization.

It is also shown that the effect is most significant for the β2ARreceptor as compared to the NK1 receptor.

BRET Assay

Cell Preparation for BRET Assay:

-   -   1. 48 hrs after transfection with 2 μg receptor-Rluc DNA (e.g.        NK1-Rluc or β2AR-Rluc) and 5 μg β-arrestin-2 DNA (e.g. the        R393E; R395E mutant) (for assays with GRK also 2 μg GRK DNA,        e.g. GRK-5) media was removed and cell washed 1× with PBS    -   2. 1 ml 1× trypsin (T75 flask) was added and incubated 3-5 min        at 37 C    -   3. 10 mL complete media was added.    -   4. Cells were transferred to tube and spun down (5 min, 800 rpm)    -   5. Media was removed, and cells resuspended in 10 ml PBS and        counted, spun down (5 min, 800 rpm)    -   6. Resuspended in D-PBS with 1000 mg/l L-Glucose (#14287) to a        density of 2×10⁶ cells/ml    -   7. Cells were left at room temperature for at least 30 min, to        stabilize readings.

Assay:

-   -   1. Dilution of DeepBlueC to 100 μM in 40% ethanol in D-PBS with        1000 mg/l L-Glucose (#14287) (light sensitive!!). The DeepBlueC™        stock solution comprises 1 mM DeepBlueC™ in 100% v/v EtOH.

The dilution of DeepBlueC in 40% ethanol/buffer is essential for thestability of DeepBlueC. If DeepBlueC is diluted in buffer alone, it willprecipitate within 10 to 20 minutes, making it virtually impossible touse the assay in HTS mode. However, when diluted in 40% ethanolDeepBlueC will be stable for hours. FIG. 3 shows the light emission fromrenilla luciferase after addition of DeepBlueC dissolved in buffer(D-PBS with 1000 mg/l L-Glucose), buffer with 6% ethanol or buffer with40% ethanol. It can be seen that the signal from the experiment using40% ethanol is stable for the duration of the experiment. On the otherhand the signals from experiments using 6% or 0% ethanol rapidlydecreases.

-   -   2. 100 μl of resuspended cells were transferred into wells in        96-well white optiplate plate    -   3. 12 μl agonist was added.    -   4. 5 μl of diluted DeepBlueC/well was added, and the plate was        read.

Antagonist:

-   -   1. Dilution of DeepBlueC to 100 μM in 40% ethanol in D-PBS with        1000 mg/l L-Glucose (#14287) (light sensitive!!)    -   2. 100 μl of resuspended cells was transferred into wells in        96-well white optiplate plate    -   3. 14 μl antagonist was added (wait 5 min.)    -   4. 14 μl agonist was added.    -   5. 6 μl of diluted DeepBlueC/well was added, and the plate read.

FIG. 2 shows the specific BRET ratio of the NK1 and the β2AR receptorscoexpressed with WT and the three different P-arrestin mutants: humanβ-arrestin-2 R393E; R395E mutant, human β-arrestin-2 373 stop mutant orhuman β-arrestin-2 R169E mutant. It is seen that the human β-arrestin-2R393E; R395E mutant and the human β-arrestin-2 373 stop mutant areincreasing the BRET signal significantly for the β2AR receptor, whereasthe effect is less pronounced for the NK1 receptor. The observed resultsare expected since the NK1 receptor is a class B receptor and the β2ARreceptor is a class A receptor.

1-35. (canceled)
 36. An assay comprising i) adding a substrate to a cellcomprising GPCR-Rluc fusion protein and a β-arrestin-GFP fusion protein,wherein the β arrestin is mutated, ii) adding a ligand to the admixtureof i), and iii) measuring a BRET signal to obtain a BRET ratio, whereinan increased BRET ratio is provided compared with the ratios obtained byuse of the same process employing a β-arrestin-GFP fusion proteinwherein the β-arrestin is the wild type β-arrestin, or employing aβ-arrestin-GFP fusion protein, wherein the β-arrestin is a β-arrestinspecifically mutated so that it acts on the receptor independent of thereceptors phosphorylation state.
 37. The assay of claim 36 wherein theligand addition can provide a GPCR-Rluc/β-arrestin-GFP complex.
 38. Theassay of claim 37 wherein separation of β-arrestin-GFP fromGPCR-Rluc/β-arrestin-GFP complex is delayed and/or inhibited.
 39. Theassay of claim 37 wherein internalization of the GPCRRluc/β-arrestin-GFPcomplex is inhibited.
 40. The assay of claim 36 wherein β-arrestin ismutated so that its binding to clathrin and/or AP2 is impaired.
 41. Theassay of claim 36 wherein β-arrestin is truncated so that it does notcontain any clathrin and/or AP2 binding sites.
 42. The assay of claim 36wherein β-arrestin is mutated by deletion, insertion or substitution sothat one or more AP2 binding sites are impaired in their binding to AP2.43. The assay of claim 36 wherein β-arrestin is mutated so that itsbinding to phosphoinositide is impaired.
 44. The assay of claim 36wherein the cell comprises a further amount of G-protein coupledreceptor kinase (GRK) as compared to the amount of GRK naturally presentin the cell.
 45. The assay of claim 44 wherein the G-protein coupledreceptor kinase is GRK
 2. 46. The assay of claim 44 wherein theG-protein coupled receptor kinase is GRK
 5. 47. The assay of claim 36wherein β-arrestin is further mutated so that it is phosphorylationindependent.
 48. The assay of claim 36 wherein β-arrestin is originatingfrom an animal source.
 49. The assay of claim 36 wherein β-arrestin is aβ-arrestin-1 or β-arrestin-2.
 50. The assay of claim 36 wherein theβ-arrestin is a human β-arrestin-1 374 stop mutant or human β-arrestin-2373 stop mutant.
 51. The assay of claim 36 wherein the β-arrestin is ahuman β-arrestin-2 R393E; R395E mutant.
 52. The assay of claim 36wherein the β-arrestin is a human β-arrestin-2 R393A; R395A mutant. 53.The assay of claim 36 wherein the β-arrestin is human β-arrestin-2K233Q; R237Q; K251Q mutant.
 54. The assay of claim 36 for use in drugdiscovery methods.
 55. The assay of claim 36 for use in high-throughputscreening:
 56. The assay of claim 36 wherein the substrate isDeepBlueC™.
 57. The assay of claim 36 wherein the substrate is used inthe form of a solution from which no visual precipitate is formed afterstorage at room temperature for at least 30 minutes.
 58. The assay ofclaim 57 wherein the solution comprising the substrate comprises one ormore organic solvents.
 59. The assay of claim 58 wherein the one or moreorganic solvents are selected from alkanols including ethanol, propanol,isopropanol, and butanol.
 60. The assay of claim 58 wherein the solventis EtOH.
 61. The assay of claim 58 wherein the solution comprises fromabout 15% v/v EtOH to about 100 v/v % EtOH.
 62. The assay of claim 61wherein the solution comprises DeepBlueC™ in 40% v/v EtOH.
 63. Asolution comprising DeepBlueC™ and one or more organic solvents, whereinno visual precipitate is formed after storage at room temperature for atleast 30 minutes.
 64. A solution of claim 63 wherein the one or moreorganic solvents are selected from alkanols including ethanol, propanol,isopropanol, and butanol.
 65. A solution of claim 63 wherein the solventis EtOH.
 66. A solution of claim 65 comprising from about 15% v/v EtOHto about 100 v/v % EtOH.
 67. A solution according to claim 66 comprisingDeepBlueC™ in 40% v/v EtOH.
 68. A method for preparing a solution ofclaim 63, the method comprising: diluting a stock solution of DeepBlueC™in a solution comprising one or more organic solvents.
 69. The assay ofclaim 36 wherein a GPCR ligand is identified.
 70. The assay of claim 69wherein the ligand is an agonist.
 71. The assay of claim 69 wherein theligand is an antagonist.